Fluorogen Activating Peptide (FAP) Technology

Fluorogen activating peptide – FAP-tags® are a new class of small genetically encoded reporters that exhibit fluorescence only in the presence of micromolar concentrations of particular nontoxic soluble fluorogens.

FAP-tags® allow the user to turn the fluorescent signal on and off by adding or removing fluorogen, or to change the signal wavelength by substituting one fluorogen for another.

This technology is particularly useful for measuring Receptor internalization, trafficking, ligand selectivity and receptor re-sensitization. Using fluorogens with different properties allows many dynamic studies of receptor trafficking. For example, if a membrane impermeant fluorogen is used, only receptors that are at the cell surface or those that visited the cell surface while the cells were exposed to fluorogen will be visible. This is in stark contrast to standard fluorescent protein fusions where the entire population of protein is fluorescent. This is clearly demonstrated in the images below:

The top set of images are acquired with a cell expressing a transmembrane domain with a FAP tag (green fluorescent fluoromodule) on the n-terminus (outside of cell) and RFP on the inside of the cell. Images were then acquired for the FAP fluoromodule and the RFP. The FAP image shows only the molecules that have reached the surface (top, left) whereas the RFP image shows the entire population of protein regardless of location. The bottom set of images show the results if a red FAP fluoromodule and GFP are used.

Spectragenetics has developed two different types of FAPs, α and β. Experiments can be designed with both FAPs to measure receptors internalization simultaneously or an individual FAP can be used for more detailed trafficking experiments. Each FAP has a unique set of available fluorogens. The α FAP has cell impermeant, permeant, pH sensitive and extra bright available. This allows measurements of protein at the surface, total protein, internalization (1 color), and trafficking to and from the cell membrane. The βFAP has 2 spectrally distinct dyes that allow for measurement of surface protein, internalization (2 color), receptor re-sensitization and pulse/chase experiments. A chart of available fluorogens for each FAP is shown below: